The overall goal of this project is the development of novel approaches that overcome the limitations of viral vectors. While recent years have seen the first cure of a genetic disease by retroviral transduction of hematepoietic stem cells in X-SCID children, the same trial has also resulted in leukemic syndromes upon activation of an oncogene by the randomly integrated Retrovirus. Preventing similar adverse events has now become a main goal of the field of gene therapy. Among Retroviruses, Lentiviruses offer unique advantages because they can provide long-term gene expression of complex genetic structures even in non-dividing cells. However, important safety concerns and manufacturing hurdles remain: (i) contamination with a replication-competent Lentivirus (RCL), (ii) oncogenesis by random insertional mutagenesis, and (iii) the unavailability of GMP-grade, high-titer, stable lentiviral packaging cell-lines. On the basis of extensive preliminary results, this proposal will attempt to remedy these important issues. In Specific Aim 1, we will build a semi-synthetic lentiviral vector in which recombinant envelope proteins or fusiogenic peptides are added to lentiviral particles packaged in the absence of any viral envelope gene. This approach virtually eliminates the possibility of RCL contamination and greatly facilitates the design of stable lentiviral packaging cell-lines by avoiding the cytotoxicity of pseudotyping envelopes. This strategy was rendered efficient by the prior magnetization of virions by super-paramagnetic nanoparticles. In Specific Aim 2, we will test the hypothesis that lentiviral vectors can be engineered to integrate site-specifically at "non-dangerous" chromosomal sites of the human genome by substituting the lentiviral integrase with a site-specific integrase from the phiC31 bacteriophage, which has been shown to operate in human cells. In Specific Aim 3, we will apply the engineering principles of "in-vitro evolution" together with the power of lentiviral libraries and selection screens to derive mutants of the phiC31 integrase with greater intrinsic activity and integration specificity within human cells. In Specific Aim 4, we will combine the previous findings to build step-by-step under GMP conditions, a packaging cell-line that produces high-titer semi-synthetic lentiviral vectors capable of non-dangerous site-specific integration in human cells. These approaches will be ultimately evaluated for their capacity to transfer a complex beta-globin gene into murine and human hematopoietic stem cells. [unreadable] [unreadable]